Wyniki wyszukiwania

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MMP-9 activation after neuronal stimulation is a polyadenylation dependent process

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Dziembowska M., Janusz A., Romanowska E., Kaczmarek M.

Acta Neurobiologiae Experimentalis

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2009

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tom 69

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nr 3

Recent studies indicate that MMP-9 (gelatinase B that regulates pericellular environment through the cleavage of protein components of the extracellular matrix) plays a role in synaptic plasticity. Szklarc zyk et al. (2002), Konopacki et al. (2008) and Wilczynski et al. (2008) have demonstrated the presence of mRNA and protein for MMP-9 at postsynaptic sides of rat hippocampal neurons. It was also shown by Michaluk et al. (2007) that gelatinolytic activity of MMP9 increases after stimulation of rat neuronal cultures with either glutamate or bicuculine. We observed the presence of MMP-9 protein and mRNA in synaptoneurosomes, the synaptic fraction isolated from hippocampus. To detect the activity of MMP-9 we measured the cleavage of its substrate, β-dystroglycan. By the use of this readout we showed that MMP-9 is activated 5 to 10 mintes after neuronal stimulation. We postulate here that MMP-9 is translated from dendritically-localized mRNA and the protein is produced in response to synaptic stimulation. Its rapid and local translation and secretion is a polyadenylation- and local translation-dependent process.

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Molekularne mechanizmy smierci komorki glejaka, indukowanej przez cyklosporyne A

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Kaminska B., Pyrzynska B., Dziembowska M., Ciechomska I.

Działalność Naukowa PAN. Wybrane Zagadnienia

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2000

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tom 10

49-50

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3’ untranslated region polymorphisms of matrix metalloproteinase 9 and their role in schizophrenia: the role in the local translation

100%

Lepeta K., Dziembowska M., Kaczmarek L.

Acta Neurobiologiae Experimentalis

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2013

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tom 73

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nr Suppl.1

Recent studies have implicated MMP-9 in schizophrenia, in particular, Domenici et al reported highly elevated plasma levels of MMP-9 in schizophrenic patients and Rybakowski et al. demonstrated an association of MMP-9 5’UTR polymorphism -1562C/T with schizophrenia. Furthermore, Dziembowska et al. have shown that MMP-9 is locally translated in neurons in response to synaptic stimulation. Since 3’UTR plays essential role in mRNA transport to the dendrites and in its local translation, MMP-9 3’UTR polymorphisms may affect synaptic availability of the enzyme. In order to verify if SNPs affect local translation of MMP-9 or its mRNA transport we have made two types of vectors with human MMP-9 containing two 3’UTR variants as well as the inactive form of MMP-9 under human synapsin I promoter. The gene constructs enable MMP-9 protein visualization by its fusion to Venus fluorescent protein and additionally contain myristoylation sequence, which is responsible for the cell membrane docking. In result, locally translated MMP-9 at the synapse could be observed. Currently, we investigate if the polymorphism influences efficiency of MMP-9 mRNA transport and local translation under basal conditions or after stimulation. To enable MMP-9 mRNA tracking in the dendrites we will use MS2 system on living neurons under basal conditions and after stimulation of the two studied 3’UTR variants.

4

Abnormal AMPA receptor trafficking in a mouse model of fragile X syndrome

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Chojnacka M., Beroun A., Kuzniewska B., Kaczmarek L., Dziembowska M.

Acta Neurobiologiae Experimentalis

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2017

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tom 77

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nr Suppl.1

INTRODUCTION: Fragile X syndrome (FXS) a common inherited form of mental retardation and autism is caused by lack of expression of fragile X mental retardation protein (FMRP). FMRP is RNA-binding protein that regulates local translation of many synaptic proteins, including AMPA-type glutamate receptors subunits. Accumulated evidence indicate that proper rates of exocytosis and endocytosis of glutamate receptors play a key role in synaptic plasticity. However, current state of knowledge of AMPA receptor trafficking in FXS models is incomplete. AIM(S): The aim of this study was to analyze AMPA receptor trafficking in a mouse model of fragile X syndrome. METHOD(S): We used synaptoneurosomes (SN) isolated from Fmr1 KO and wild-type (WT) mice and stimulated them in vitro with NMDA/glutamate. To determine levels of surface and intracellular GluR1, GluR2 and GluR3 we used crosslinking of SN with BS3 reagent followed by western blot analysis. To confirm our biochemical results we investigated the synaptic calcium-permeable AMPA receptors using whole-cell patch-clamp recordings. RESULTS: We found that SN stimulation produced an increase in the surface glutamate receptor subunits only in WT mice. We also found that surface GluR2 protein level was significantly higher in Fmr1 KO SN in basal conditions, when compared to WT. The electrophysiological experiments confirmed higher abundance of GluR2‑containing AMPA receptors in the hippocampus of Fmr1 KO mice. CONCLUSIONS: Our results indicate that Fmr1 KO mice exhibit abnormal AMPA receptor trafficking and it is demonstrated by elevated amount GluR2.

5

Fragile X mental retardation protein regulates synaptic translation of neuroligins

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Podsiadlowska J. J., Kuzniewska B., Milek J., Urbanska K., Dziembowska M.

Acta Neurobiologiae Experimentalis

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2017

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tom 77

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nr Suppl.1

INTRODUCTION: Neuroligins (NLGNs) are postsynaptic cell adhesion proteins which bind to their presynaptic partners neurexins across the synaptic cleft. Thus, NLGNs are crucial for the formation, maturation and maintenance of synapses. In rodents, neuroligins are encoded by four genes: Nlgn1, Nlgn2, Nlgn3 and Nlgn4. The mutations in Nlgn3 and Nlgn4 genes are associated with autistic phenotype. Another cause of autistic behaviors, fragile X syndrome, results from the lack of fragile X mental retardation protein (FMRP). FMRP binds to neuronal mRNAs and regulate local translation of transcripts that play an important role in synaptic signaling and plasticity. AIM(S): We aimed to determine if synaptic translation of Nlgn1, Nlgn2 and Nlgn3 mRNAs is regulated by FMRP. METHOD(S): We used Fmr1 knock-out mice (Fmr1 KO) and their wild type (WT) littermates to isolate synaptoneurosomes, which were stimulated in vitro to induce local protein synthesis. We performed FMRP IP on synaptoneurosomes and FISH combined with FMRP immunostaining on cultured neurons to investigate Nlgns mRNAs interaction with FMRP. The polyribosome fractionation was used to elucidate if FMRP regulates Nlgns mRNAs local translation. To study the surface versus intracellular NLGNs distribution at WT and Fmr1 KO synapses we have chosen chemical crosslinking and biotinylation assays, followed by Western blotting. RESULTS: We show that mRNAs for three studied neuroligins interact directly with FMRP in synaptoneurosomes and Nlgn1, Nlgn2, Nlgn3 mRNAs colocalize with FMRP in dendritic granules of cultured hippocampal neurons. The Nlgn1, Nlgn2 and Nlgn3 mRNAs associate with translating polyribosomes in response to synaptic stimulation and Fmr1 KO mice exhibit upregulated local translation due to the lack of FMRP. Finally, the excessive local synthesis of NLGN proteins at Fmr1 KO synapses leads to their elevated level on the postsynaptic membrane. CONCLUSIONS: Nlgn1, Nlgn2 and Nlgn3 mRNAs are locally translated at the synapse and FMRP regulates this process. FINANCIAL SUPPORT: Supported by NCN grant Sonata Bis 2014/14/E/NZ3/00375.

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Control of microphtalmia transcription factor localization through regulated isoform-specifif nuclear shuttling

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Dziembowska M., Cigna N., Anezo O., Cordelieres F. P., Klein C., Saule S.

Acta Neurobiologiae Experimentalis

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2007

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tom 67

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nr 3

7

Chronic fluotexine treatment disrupts apetitively motivated learning and central amygdala structural plasticity

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Puscian A., Leski S., Winiarski M., Charzewski L., Nikolaev E., Dziembowska M., Knapska E.

Acta Neurobiologiae Experimentalis

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2015

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tom 75

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nr Supl.

AIMS: Fluoxetine, a selective serotonine reuptake inhibitor, is commonly used to treat psychiatric disorders. Available data show that fluoxetine has limited side effects and, more importantly, may improve patient’s cognitive abilities. However, little is known about the mechanisms by which fluoxetine affects learning, especially appetitively motivated one. Thus, in the present project we investigated the effects of a long-term fluoxetine treatment on appetitively motivated discrimination learning. METHODS: We used fully automated behavioral assessment of discrimination learning in group-housed subjects, DI-staining for determining changes in morphology of dendritic spines and gel zymography for measurement of activity of MMP-9 (matrix metaloproteinase 9, an enzyme involved in synaptic plasticity). RESULTS: We showed that above-described learning is severely impaired in mice subjected to the long-term fluoxetine treatment. Since we have previously shown that such learning depends on MMP-9 activity in the central amygdala (CeA), we examined MMP-9 activity in the CeA of the fluoxetine treated mice. We found decreased MMP-9 level. Further, we tested fluoxetine influence on dendritic spine morphology in the CeA and observed that behavioral performance of the control wild type mice was highly correlated with a size and of mature, mushroom-shaped dendritic spines. No such correlation was found in MMP-9 knock out mice. Applied treatment abolished this correlation in wild type mice and did not reinstated it to a significant level in MMP-9 knock outs. CONCLUSIONS: Obtained results show that chronic fluoxetine treatment impairs appetitive discrimination learning in healthy controls, decreases MMP-9 activity and disrupts correlation between subjects’ performance in appetitive learning and structural synaptic plasticity in the CeA. The data shed light on dendritic spines’ dependent learning mechanisms, that may be disarrayed in the CeA by commonly applied fluoxetine treatment in patients.

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MiR-132 regulates MMP-9 protein levels in vivo by targeting its 3’UTR region

76%

Kuzniewska B., Gewartowska O., Jasinska M., Gruchota J., Rejmak K., Dziembowski A., Dziembowska M.

Acta Neurobiologiae Experimentalis

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2017

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tom 77

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nr Suppl.1

INTRODUCTION: MicroRNAs (miRNAs) are small noncoding RNAs that bind to target sites in mRNAs, leading to translational repression. MiRNAs are present in dendrites and synapses where they are believed to fine‑tune the local expression of synaptic proteins. MiR-132 is a neuronal activity-regulated microRNA that controls the morphology of dendritic spines and synaptic transmission. Similar activities have recently been attributed to matrix metalloproteinase-9 (MMP-9), an extrasynaptic protease. Our previous studies show that miR-132 can directly regulate Mmp-9 mRNA by targeting its 3’UTR in cultured primary neurons. AIM(S): In the current study, we aimed at verification whether miR-132 regulates the expression of Mmp-9 in vivo in the mouse brain. METHOD(S): To determine whether miR-132 binds to the 3’UTR of Mmp-9 mRNA, the luciferase reporter assay using the coding sequence of firefly luciferase fused with the 3’UTR of Mmp-9 mRNA. Next, CRISP-Cas9 technology was used, in order to introduce mutations in putative binding site for miR-132 in 3’UTR of Mmp-9 locus in mice. Subsequently, gelatin zymography was used to evaluate the levels of MMP‑9 protein in different brain regions of mutant and control mice. RESULTS: Overexpression of miR-132 in cortical neurons significantly reduced the luciferase activity of MMP‑9 3’UTR reporter. Importantly, miR-132 failed to regulate the mutated MMP‑9 3’UTR luciferase reporter, confirming the functionality of the predicted sequence within the 3’UTR of MMP-9. Mutation in 3’UTR region of MMP-9 targeted by miR-132 in mice, resulted in higher MMP-9 protein levels in different brain regions of mutant mice as compared to controls. CONCLUSIONS: We show, that miR-132 binds to the 3’UTR of Mmp-9 mRNA in primary cortical neurons. Moreover, we developed a new mouse model to study miR-132 – Mmp-9 interaction in vivo. Our data suggest, that miR-132 targets 3’UTR of Mmp-9 mRNA in vivo and can regulate MMP-9 protein in mouse brain. FINANCIAL SUPPORT: Supported by NCN OPUS 2014/15/B/NZ3/01054.

Ograniczanie wyników

MMP-9 activation after neuronal stimulation is a polyadenylation dependent process

Molekularne mechanizmy smierci komorki glejaka, indukowanej przez cyklosporyne A

3’ untranslated region polymorphisms of matrix metalloproteinase 9 and their role in schizophrenia: the role in the local translation

Abnormal AMPA receptor trafficking in a mouse model of fragile X syndrome

Fragile X mental retardation protein regulates synaptic translation of neuroligins

Control of microphtalmia transcription factor localization through regulated isoform-specifif nuclear shuttling

Chronic fluotexine treatment disrupts apetitively motivated learning and central amygdala structural plasticity

MiR-132 regulates MMP-9 protein levels in vivo by targeting its 3’UTR region